The protease factor Xla (fXIa) contributes to hemostasis by sustaining thrombin generation after initiation of blood coagulation. In addition to contributing to fibrin formation, fXIa promotes clot resistance to fibrinolysis in a process involving the proteinase TAFI. Several lines of evidence indicate fXIa contributes to pathologic coagulation. Elevated plasma levels of the fXIa precursor fXI are associated with increased risks for arterial and venous thrombosis in humans. We observed that fXI deficient mice are resistant to vascular occlusion in arterial injury models, indicating fXIa contributes to platelet rich thrombus formation. FXI binds to platelets in vitro, and is converted to fXIa by thrombin or factor XI la (fXlla). The importance of these interactions in vivo or in complex in vitro systems is not known, and the relative importance of thrombin and fXlla to fXI activation is not clear. FXI I deficiency does not cause a bleeding abnormality, indicating fXI is activated in vivo by other proteases. However, fXII deficient mice are resistant to arterial occlusion, raising the possibility that formation of a pathologic occlusive thrombus involves processes distinct from those involved in normal hemostasis. In Aim 1 of this proposal, we will use mice with deficiencies of fXI, fXII, and TAFI to investigate the importance of these proteins in models of pathologic coagulation. Using a panel of novel recombinant fXI variants to reconstitute fXI deficient mice, we will determine the importance of fXI interactions with platelets, thrombin and fXlla to these models, and look for alternative functions for fXI and fXII unrelated to classical coagulation pathways. In Aim 2, a murine fXI-dependent bleeding model will be used to determine if fXI interactions with platelets, thrombin and fXlla are required for hemostasis. In Aim 3 we will use human and murine platelet rich plasma systems to examine the importance of fXI and fXII to thrombin generation and platelet activation in vitro, with the primary goal of determining if there are differences between the two species. These studies will allow us to compare the importance of fXI, fXII and TAFI to normal hemostasis and to pathologic coagulation, and will provide valuable information regarding the mechanism(s) for fXI activation in complex systems. Ultimately, the work may demonstrate that fXI and/or fXII contribute in a substantive way to blood vessel occlusion, and are therefore, legitimate targets for novel investigative therapies to treat or prevent vascular thromboembolism in humans. Relevance: Anticoagulant drugs have been very beneficial for treating abnormal blood clots in a variety of diseases, but have the disadvantage of leaving the patient prone to serious bleeding. We are looking for new targets in blood for anticoagulation therapy that will not be associated with high bleeding risk. [unreadable] [unreadable] [unreadable]